JPH03224478A - Biologically pure culture fluid for microor- ganism and preparation of lactone, diol, compound and cyclic ether - Google Patents

Abstract

PURPOSE: To produce sclareolide and a diol from a mixture of sclareol with episclareol by culturing a specific microorganism in the subject culture solution.

CONSTITUTION: This method for producing sclareolide comprises culturing Cryptococcus albidus, ATCC 20918, or Cryptococcus albidus, ATCC 20912, in an aqueous nutritive culture medium under an aeration condition. The method for producing a diol of the formula comprises culturing Bensingtonia ciliata, ATCC 20919, or Cryptococcus laurentii, ATCC 20920, in the above culture medium under aeration.

COPYRIGHT: (C)1991,JPO

Description

[Detailed description of the invention] [Summary of the invention] The present invention Biologically pure cultures of the following microorganisms: It is related to.

Cryptococcus albidus.

ATCC20918゜Bensingtonia ciliata, AT
CC20919, Cryptococcus 1au
renLii, ATCC20920, and Cryp
tococcus albidus, ATCC209
21 In another embodiment, the present invention relates to a culture solution for the following microorganisms.

Cryptococcus albidus, ATC
C20918, Bensingtonia cilia
La, ATCC20919, Cryptococcu
S 1aurentii, ATCC20920, and Cryptococcus albidus, ATC
C20921 Each of these cultures can produce diols having the following structure or either of the following in an aqueous nutrient medium under aerated conditions as follows.

Cryptococcus albidus, ATC
C20918 and Cryptoc.

ccus albidus, ATCC20921,
Sclareol with the following structure [sc 1area l]
and the following structure of episclareol [episclare
ol] can also be produced from a mixture with Bensingtonia ciliaLa.

ATCC20919 and Cryptococcus laurentii.

ATCC20920 specifies sclareol [5cla reol] with the following structure and episclareol [episclareo] with the following structure.
l] It is possible to produce diols having the following structure.

In yet another embodiment, the invention also relates to a mixture produced by culturing (individually) the following microorganisms under aerated conditions in an aqueous nutrient medium:

ATCC20918, Cryptococcus al.
bidus.

ATCC20919, Bensingtonia ci
liata.

ATCC20920, Cryptococcus 1
aurentii.

ATCC20921, Cryptococcus al.
In yet another embodiment, the present invention provides the following methods: (1) Cryptococo of ATCC 20918 under aerated conditions in an aqueous nutrient medium containing one or more compounds of the following structure and/or the following structure:
ccusalbidus or ATCC20921 C
The present invention relates to a method for producing sclareolide having the following structure, which is characterized by culturing any microorganism such as ryptococcus albidus.

In yet another embodiment, the invention relates to a process for the production of a product having the following structure: (a) ATCC 20919, Bensington
a ciliata, or (b) ATCC2092
0, Cryptococcus 1aurentii, and one or more compounds selected from the group consisting of (1) sclareol having the following structure, (2) evisclareol having the following structure, and (3) acetate having the following structure. It is characterized by culturing in an aqueous nutrient medium with aeration conditions.

The process of converting compounds is performed by converting Cryptococcus al.
bidus.

ATCC20919 Bensingtonia ci
liata.

Cryptococcus 1a from ATCC20920
urentii, or Cryp of ATCC20921
A microorganism such as tococcus albidus
This includes the cultivation of

These compounds can thus be used alone or as a mixture containing any number of the above-mentioned compounds.

Thus ATCC20918's Cryptococcu
s albidus.

or ATCC20921 Cryptococcus
The following reaction can be carried out using albidus.

and/or ATCC20919 Bensingtonia ci
liata, or ATcc 20920 Crypt
When the reaction is performed using ococcus 1aurentii, the following reaction can occur.

and/or The form of the microorganism used is not important.

The microorganism can be used as a culture medium (suspension) containing the cells and a suitable nutrient solution for the cells, or in the form of cells suspended in a buffer. Cells or their extracted enzymes can be immobilized on a suitable solid support and they can then be used for the conversion of compounds.

A suspension culture mixture is prepared by inoculating a suitable aqueous nutrient medium with the microorganism. A suitable nutrient medium includes a nitrogen source, inorganic salts, growth factors, a suitable substrate, and optionally other carbon sources. Types of carbon sources suitable for carrying out the method of the invention include, for example, glucose, galactose, L-sorbose, maltose, sucrose, heptalobiose, trehalose, L-arabinose, L-rhamnose, ethanol, glycerol, L-distritol, D-mannitol, lactose, melibiose,
Raffinose, meletitose, starch, D-xylose, D-sorbitol, a-methyl-D-glucoside,
There are lactic acid, citric acid, and succinic acid. Suitable nitrogen sources include, for example, nitrogen-containing organic substances such as peptone, meat extract, extracted yeast, cornstarch liquor, casein, urea, amino acids, or nitrogen-containing organic substances such as nitrates, nitrites, inorganic ammonia salts. There are inorganic compounds. Suitable inorganic salts include, for example, magnesium, potassium, calcium, or sodium phosphates. The nutrients in the above-mentioned medium may include, if desired, for example, 1 or 2 or more of vitamin B group;
and/or Fe, Mo, Cu.

It can be supplemented with one or more trace minerals such as Mn and B. Vitamins and trace minerals are unnecessary when small amounts of yeast extract are added to the medium. When bacterial contamination is a problem, use chloroamphenicol [chlo
It is preferred to add an antibiotic such as roamphenicol] or chlorotetracycline.

Cultivation of microorganisms can be carried out either as static culture or in deep culture under aerated conditions (e.g., stirred culture, fermenter [fermentor]).
It can also be carried out as a culture using [Entor]. Depending on the situation, the pH may range from about 2.5 to 9.0°, preferably from about 3.0 to 7.5, most preferably from about 3.0 to 6.5. pH value is hydrochloric acid,
It can be prepared by adding inorganic or organic acids such as acetic acid, oxalic acid, or by adding bases such as sodium hydroxide, ammonium hydroxide, or phosphates or phthalates. This can be adjusted by adding a suitable buffer. The incubation temperature should be maintained at about 12°C to about 33°C, more preferably about 15°C to 30°C, optimally about 18°C to 28°C.

The method according to the present invention is suitably carried out by adding one or a mixture of compounds and/or of the following structure as a single carbon source to a nutrient medium at the beginning of culture. Alternatively, the substrate may be added in combination with another carbon source, such as dextrose, during cultivation or when the carbon source is depleted. The only limitation on the substrate concentration in the culture medium is the ability to effectively aerate the culture. However, the substrate concentration is preferably between about 0.1 g/Q and about 1
30g/Q, about 0.5g/12-120
g/Q.

Optimally, the range is from about 2.5 g/Q to about 100 g/ff. Conversion of compounds may be optimally carried out under any of the circumstances described above.

The total time for compound conversion (after the initial incubation period) will vary depending on the composition of the nutrient medium and substrate concentration.

In general, culture using a shaking 7-laser is about 12 hours to about 26 hours.
It takes 4 hours. However, when fermenters are used, the culture time is reduced to about 48 hours or less.

Conversion of the compounds is carried out using microbial cells isolated from the culture medium or extracted enzymes isolated from the cells by methods well known in the art. In this case, the conversion of the compounds is conveniently carried out in various aqueous nutrient media, for example in buffered solutions, in saline solutions, in fresh nutrient media or in water. The isolated cells or extracted enzymes are immobilized on a solid support to accomplish the conversion of the desired compound.

The conversion of substrate compounds is also influenced by mutants of this organism (microorganism). Such mutants can be easily obtained by well-known methods, eg by exposing the cells to ultraviolet light or X-rays, or to known mutagens, such as eg acridine orange.

The substrate is available as a powder or as TWEEN 80(TM)
as a slurry in an emulsifier such as (polyoxyethylene sorbitan monooleate) or as a solution in an emulsifier or in a hydrophilic solvent such as acetone, methane, ethanol, ethylene chloride or dioxane. It can be added to the culture medium as a solution in the medium. Surfactants or dispersants can also be added to the aqueous suspension of the substrate, or the substrate can be emulsified using ultrasound.

Commonly available antifoam agents such as silicone oil (eg, UCON), polyalkylene glycol derivatives, corn oil, or soybean oil can be used to suppress foaming.

Substrate conversion is by gas-liquid chromatography (GLC).

It can be monitored using standard analytical methods such as thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), infrared spectroscopy (IR), and nuclear magnetic resonance (NMR). If rapid disappearance of substrate is observed, more substrate can then be added to maximize the ability of the microorganism to convert the compound. This process generally resulted in the disappearance of most of the substrate from the medium. Sometimes it ends.

Depending on the type of microorganism used, under Or a compound with the following structure S Can be recovered from aqueous nutrient media.

The compound having the following structure is described in No. 81!4 of U.S. Pat. No. 4,798,799,
As described in lines 52 and 53 (which is incorporated herein by reference), it is cyclized to a compound having the following structure.

Also, It may be used because of its value as a fragrance. Alright is a compound with the following structure It may be reduced through the following reaction.

The formation of a compound with the following structure is First, a compound with the following structure reduced to a diol with the following structure, The diol is then re-closed, This is done by producing a compound with the following structure.

This is due to the following reaction.

and preferably this reduction reaction is carried out using a reducing agent such as VITRIDE™ - hydrogenated sodium bis(2-methoxyethoxy)aluminum hydroxide as described in U.S. Pat. No. 3,507,895. It is carried out in the presence of an inert solvent such as toluene using moo. This reaction is preferably carried out at about 0°C.
It is carried out in a temperature range of ~90°C for about 1 hour to about 3 hours.

The reclosing reaction, i.e., is preferably carried out under basic conditions, for example in an aqueous alkali metal hydroxide, such as aqueous potassium hydroxide or aqueous sodium hydroxide. The details will be described in Example (2) below.

Isolation and purification of compounds of the structure below from fermentation broth can be carried out by conventional techniques such as filtration, centrifugation, solvent extraction, distillation, crystallization, etc.

Compounds having the following structure are described in U.S. Pat. No. 4,798,799, column 8, 58-6.
Line 8, column 9 of U.S. Pat. No. 4,798,799, No. 1
It can be converted into a compound of the following structure by a commonly used cyclization method well known to those skilled in the art described in lines 2 to 2.

Each microorganism used in the present invention was isolated from soil samples obtained from various geographical locations.

Each strain was deposited at the American Type Culture Collection [ATCC] under the following accession number.

Cryptococcus albidus, ATC
C20918: Bensingtonia cilia
ta, ATCC20919; Cryptococcu
s 1aurentii, ATCC20920;
and Cryptococcus albidus, ATC
C20921Bensingtonia ciliaL
a and Cryptococcus 1aurentii
Both microorganisms were studied at the Central Bureau Vaucimer Cultures (CBS), which gave them the following names: That is, Lecythophere hoffmannii (
van Beijma), W, Gam5 (synon
ym Ph1alophora hoffmannii
) because, according to CBS, it is a filamentous fungus.

Cryptococcus albidus, ATC
C20918 was also examined at CBS, which identified this culture as Cryptococcus albidus var.
, albidus (Saito) 5kinner
It was named.

Cryptococcus albidus, ATC
C20918 is described as follows.

Morphology: Growth in liquid medium revealed unipolar budding cells. A thin film appeared on the surface of the liquid, while heavy sediments were observed. Growth on solid agar was unicellular, white to slightly pinkish, very shiny, bright, sticky, with round, well-circumscribed colonies. pseudohyphael
were not formed on cornmeal agar.

Physiology and Biochemistry: Carbon fixation: Carbon fixation: (Proliferation) (Proliferation) Glucose + D-Lipose Unknown Galactose + L-Rhamnose + L-Sorbose +
D-glucosamine + maltose + ethanol unknown sucrose + erythritol cellobiose + glycerol unknown trehalose + adonitol (ribitol) + lactose + dulcitol (galactitol) + melibiose D-mannitol 10 raffinose + D-sorbitol (dulcitol) + meletitose + a-methyl-D - Glucosynulin Soluble starch D - Xylose L - Arabinose D - Arabinost + Salicin inositol Lactic acid Citrate Succinic acid Growth at 30°C + Growth at 37°C Growth without vitamins Arbutin splitting + Nitrogen assimilation: NH, N03 KNO.

No.

Ethylamine fermentation (acid formation) glucose galactose maltose sucrose lactose raffinose melibiose inulin cellobiose meletitose starch trehalose Bensingtonia ciliata, ATC
C20919 is described as follows.

Morphology 2 Yeast maintenance broth (ATCC medium #200)
Above, the cells are spherical with 1-3 buds per cell.

In solid medium the cells become filamentous with extruded spores produced. Colonies are salmon yellow-brown in color, flat and overcast, with distinct borders. True mycelium was observed after 3 weeks on cornmeal agar (ATCC medium #307).

Physiology: Carbon fixation nigercose + D-ribose galactose
+ L-Rhamnose + L-Rhamnose +
D-glucosamine + maltose + ethanol weak sucrose + erythritol + cellobiose + glycerol + trehalose + adonitol (ribitol) weak lactose sulcitol (galactitol) weak melibiose + D-mannitol weak raffinose + D-sorbitol (dulcitol) weak meletitose + a -Methyl-D-glucoside Weak inulin Soluble starch D-xylose -Arabinose + D-arabinose + + Salicin inositol Lactic acid Citrate Succinic acid Weak Growth without vitamins Nitrogen assimilation: NH, NO, growth at mild high temperatures: a little less than 30°C / 37℃ Taxonomic description: Bensingtonia ciliata C, T,
Ingold (see @ below) Line drawing style (Fungi Imparfe)
ctil ejected spore fungi (spores that are blown away with force) The ejected spores are colorless and oval in shape, measuring 2 x 5 µm in liquid medium, often 8 x 5 µm, with a pointed tip and a flattened base.

Ejected spores form yeast-like budding spores and germinate. The above-mentioned budding spores form a typical yeast colony while repeating sporulation (see photo).

Some types of extruded spores germinate with the formation of short hyphae that produce extruded spores and the repeated release of spores, resulting in an overall hyphal-type colony (see photo).

Evaluation from the following media: ATCC medium #307''-Nmeal agar (Dirco 038
6) and scholastic cornmeal agar, yeast malt agar (Dirco 0712) Neurospora agar (Difc.

0321) EPD Malt Extract Agar (Difco 0024) Potato Dextrose Agar #200 #324 #1245 #331 #336 #343 V-8 Youth Agar @ 1. Ingold, C.T. (1986)
Bensingtonia ciliata Gen,
et, sp, nov,, Ba1listopo
ric fungus.

Trans, Br, Mycol, Soc, 86 (
2): 325-3282, 1ngold, C, T,
(1988) Bensingtonia cili
Further observations regarding ata Trans, Br,
Mycol, Soc, 91(1): 162-16
6Cryptococcus 1aurentii,
Regarding ATCC20920, it is described as follows. Namely, Physiology and Biochemistry: Carbon Fixation: (Growth) Glucose + Galactose + L-Sorbose D-Glucosamine Maltose + Sucrose + Cellobiose + D-Lipose Rhamnose + Variable Ethanol + Erythritol + Glycerol + Trehalose + Adonitol (ribitol) Dogu lactose Juyao dulcitol (galactitol) Variable melibiose
+ D-mannitol + raffinose D-sorbitol (dulcitol) + meletitose + a-methyl-D-gluco-do + inulin + chelicin soluble starch + inositol D-xylose + lactic acid L-arabinose + citric acid di-arabinose 10 weak succinic acid Nitrogen assimilation diNH, NO.

KNO.

O2 Ethylamine Growth without vitamin + Arbutin fission + Fermentation (gas production) Glucose Galactose Maltose Sucrose Lactose Raffinose Melibiose Inulin Cellobiose Meletitose Starch Trehalose Morphology: Pink slimy colonies, round germinated cells, in flask heavy precipitate; thin mycelium formed on the Dalmau dish.

Reference: C.P., Kurtzman, Mycolo
gia 65; p, 388-395.1973 Cryptococcus albidus, ATC
C20921 is described as follows.

Physiology and Biochemistry: Carbon fixation: (growth) Glucose + D-ribose galactose + L-rhamnose + L-sorbose + D-glucosamine + maltose
+ ethanol variable sucrose + urithritol cellobiose glycerol variable trehalose + adonitol (ribitol) variable lactose dulcitol (galactitol) + melibiose D-mannitol + raffinose + D-sorbitol (glucitol) meletitose + a-methyl-D-glucoside inulin soluble Starch + D-xylose + L-arabinose + D-arabinose + salicinositol lactic acid citrate succinate + + Nitrogen assimilation: NH, NO.

KNO.

O2 Ethylamine Growth without vitamin + Arbutin fission + Fermentation (gas production): Glucose Galactose Maltose Sucrose Lactose Raffinose Melibiose Inulin Cellobiose Meletitose Starch Trehalose Morphology: Yellow-brown, slimy colonies. Round germinated cells. Heavy sediment, pseudomycelium or true mycelium in the flask.

[Example Figure 1 shows Cryptococcus albidus
(ATCC20918) is a photograph of the thin layer chromatography development spot of the product of the example process at pH when using 1) sclareolide with the following structure, 1 diol mountain body with the following structure, and sclareol (with the following structure). (substrate).

Li workman Figure 2 shows GC-MS of starting material of Example ① It is a spectrum.

The peak indicated by the symbol 21 is The peak indicated by the symbol 20 is A compound with the following structure, That is, it is the peak of the internal standard compound.

FIG. 3 is a nuclear magnetic resonance [NMR] spectrum of the reaction product of Example 2, which is a compound having the following structure.

S Figure 4 shows The reaction product of Example ■ having the following structure It is Kutle.

The following examples are described to show specific examples of the present invention that are currently preferred, and are not intended to limit the scope of the present invention in any way. Unless otherwise specified, weight is in Durham, temperature is in Celsius, and pressure is in mm/Hg.

(Sait.

Example I Cryt+tococcus albidus [5ki
nner var, albLdusl) (Effect of pH when converting sclareol to sclareolide using ATCC 20918) Reaction: and the following media were prepared.

N H4N O30-1% KH, PO40, 1% Mg5O, 7H, OO, 05% Yeast extract 0.2% Each loO + nQ medium and 1 g sclareol in TWEEN80 (TWEEN80: sclareol = 2 = 1 ) containing 11 flasks. Each flask was inoculated with 5mα of a 24-hour culture grown on dextrose at 25°C and 150 rpm. Products and substrates were monitored by thin layer chromatography against known standardized gold standards.

Here, "substrate" means A compound with the following structure, Compounds with the following structure 80= Sclareol, a mixture of 20 say.

"Intermediate" What is Refers to a compound with the following structure.

What is “product”? Sucra, a compound with the structure below leolai It says "do".

Table 1 Flask Hour number p) (24 hours 1 2.5 TP+S+TI2
3.0 P+TS+13 3.5 P+
TS+TI 4 4.0 P+TS41 5 4.5 P+TS+1 6 5.0 P+TS+1 7 7.0 P+S+1 8 7.5 P+S+1 9 8.0 P+S+1 10 8.5 P+S+1 11. 9. OP+S+r [Strain] TS: Trace substrate S: 48 hours P+S+ T p+s+ r P+Tl P+Tl P+1 P+1 P+1 P+1 P+TI+I P+TS+ I P+TS+1 Substrate TP-72 hours P+S+TI P+Tl P+T I P+Tl P+1 P+1 P+1 P+1 P+1 Trace product P: Product I : Intermediate T and trace intermediate figure 1 shows Cr
yptococcus albidus (ATCC2
0918) is a spot of thin layer chromatography eluate of the product.

EXAMPLE ■ During the production reaction of diol intermediates and screening of 10 soil samples brought in from Greenwood 7/st, Barnegat Township, New Jersey, several flasks were Thin layer chromatography showed spots corresponding to compounds with the following structure.

The following media were prepared.

NH, NO30, 2% KH2F0. 0.1% MgS○4-7H200.05% Yeast extract 0.2% Dextrose 1.0% 5. In an OOmQ flask, 100 m+2 medium and Sucra 1/ol powder: TWEEN 80 (trademark) 5.
1 g of a 0:50 mixture was added. This flask was produced by Bensingtonia ciliata,
It was inoculated with 400 microliters of isolate ATCC20919. After one week at 25℃ 115Q rpm,
The resulting product was extracted with 330 mQ of ethyl acetate and the extract was dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator. The residue was dissolved in warm hexane and ethyl acetate. The extract was naturally evaporated over 24 hours, when pure crystals of the compound with the following structure (
350 mg) was obtained.

FIG. 3 is a nuclear magnetic resonance spectrum of a compound having the following structure.

Example ■ Cryptococcus albidus (Sait
o [5kinner var, albidus])
, Sclareolide production reaction using ATCo 20918: and medium NH, NO, 0.2% KH2P0. 0.1% Mg5O,.7H200.05% Extracted yeast 0.2% Antifoaming agent 10.0g d-H2O8.5ff Fermented body parameters Temperature: 25°C Aeration: ], OQ/min.

Stirring: 430 rpm) (25%
Controlled by NaOH to 5,8.

Duration = 4 days Substrate preparation: 500g sclareol, 250g TWEEN 8
0 and 11259 water were added to a blender and stirred for 5 minutes to form an emulsion.

A fermenter containing the above medium was sterilized at 121°C for 30 minutes and cooled to 25°C. This fermenter is Cryptoc
occus albidus (Saito [5ki
nner var, albidus]) ATCC
20918 was inoculated with 300 mQ of 24-hour growth bacteria.

600 g of sclareol emulsion was added at the time of inoculation and 6009 emulsion was added at 24, 48 and 72 hours. After 96 hours, the contents of the fermenter were filtered through a 400 mesh sieve. The crude product thus obtained is dissolved in IPA,
When filtered and the solution is concentrated, it crystallizes and produces 43
0 g of pure sclareolide was obtained.

Figure 2 shows GC-MS of the initial reaction mixture in this example
It is a spectrum. The peak designated by numeral 21 is the peak of sclareol, which is an 80:20 mixture of compounds having the following structure.

The peak indicated by the symbol 20 is the peak of the internal standard compound of the compound having the following structure.

Figure 4 shows Generated by this example ■ with the following structure Sclareolai Nuclear magnetic resonance spectrum of Tor It is.

Example ■ Cryptococcus albidus.

Example of production of sclareolide from sclareol using ATCC20918 ■
The same media and parameters were used as those used in . However, the method of substrate preparation and addition was changed.

160 g of sclareol powder and 80 g of TWEEN 80™ were added to the medium prior to sterilization. An alternative substrate was prepared by mixing finely divided sclareol (2 parts) and TWEEN 80 (1 part) to form a paste. 225g portions of this paste were added at 24, 48 and 72 hours after inoculation.

A total of 655 g of crude sclareolide with a purity of 67.34% was obtained.

Example V Cryptococcus albidus, ATC
Production of sclareolide from sclareol using C20921 The same medium and parameters as used in Example 3 were used. However, the amount of substrate and agitation were varied. .

150g of sclareol and TWEEN80 (trademark) 7
59 was added to the medium prior to sterilization.

Only 241 g of pasty substrate was added 24 hours after inoculation. Stirring was started at 430 rpm and then at 6
Increased to 30 rpm.

A total of 491 g of crude sclareolide with a purity of 44% (
A molar conversion of 94.7%) was obtained.

Example ■ sclareonopediol intermediate, and from diol acetate Production of sclareolide reaction: and The following media were prepared.

N　H4N　Ox KH! P04 M g S O4・7N (20 extracted yeast 0.2% 0.1% 0.05% 0.2% Dextrose 0.5% A 10 (1 volume) fermenter was used with the following operating conditions.

Temperature = 25°C pH: 6.0 Agitation: 430 rpm Sterilization: 30 minutes at 121'C The fermentor was inoculated with a 48 hour shake flask culture lOOmQ grown in the same medium at 25°C and 150 rpm.

After 24 hours of multiplication, each issue in the issuer is 200
l O,0 in a cooled centrifuge.
Centrifuged at 0 rpm for 0 minutes. The cells in each tube were washed twice with Butterfield's phosphate-buffer.

Buffer Preparation Storage Solution Monopotassium Hydrogen Diphosphate 34.09 Distilled Water
500.0m (2 approx. 175m12 1.ON
(Regular) Adjust the pH to 7.2 with sodium hydroxide solution,
Dilute to IQ and store.

Diluent: Dilute 1.25 mQ of stock solution to 1Q with distilled water. Prepare diluted blind solution in a suitable container. 15 at 121℃
Sterilize for minutes.

The cells are then harvested into 100 mQ of this buffer.

The pH was adjusted to 6 and the mixture was transferred to a 500m0 flask.

Compounds tested: a, compound 1 - sclareol in TWEEN80 (
1:2) Paste b, compound 2 - acetate with the following structure at TWEEN80
A 300NO2 flask containing 100 ml of buffer and cells (resting cells) were heated at 25°C and 150 rpm. Samples were placed in a shaker incubator at 24 hours, 48 hours, and 72 hours and analyzed against standard known compounds using thin layer chromatography.

In the following table (Table ■), P is the product sclareolide with the following structure; S is the substrate with the following structure in a ratio of one compound: 80:20; and I is the compound with the following structure. Intermediate: Table ■ Flask Period number Substrate 24 hours 1 Compound I P+S+1 2 Compound 2 P+TS+1 3 Compound 3 7P+S (added 1 g of each) Interval 48 hours 72 hours P+I P+I P+I P+TI P+S P+TS Example ■ Bensingtonia ciliata, A T.C.
Production of diol intermediate from sclareolide using C20919 An experiment was conducted in the same manner as in Example ① with the following specifications.

Microorganism: Bensingtonia ciliata
, ATCC20919 medium: NH<NO340-Og Extracted yeast 40.0gKH2POt
20. OgMgSO,,7H,010
,Og Sclareol 160.0gTWEEN 8
0 80 , Ogd-H,o
19. OQ Fermentor Parameters: Temperature: 25°C Aeration = 2Q/min Stirring: 300rpm pH: Controlled to 6.0 with 25% NaOH Period: 15 days The fermenter with the above medium was sterilized at 121°C for 30 min, then sterilized at 25°C. It was cooled to This fermenter was grown for 48 hours on Bensingtonia ciliata, AT
It was inoculated with CC20919 culture IQ. After 15 days of incubation, the substrate had become the product.

The contents of the fermenter were filtered through a 400 mesh sieve. The crude product thus obtained was air-dried. 101.7 g of product was obtained.

EXAMPLE ■ Preparation of decahydro 3a,6,6,9a-tetramethylnaphtho[2b cotran and 124 mQ VI of
TRIDE™ (sodium bis(2-methoxyethoxy)aluminum hydride) was added.

500+nQ of toluene was added to the VITRIDE without stirring. 50 g of sclareolide of the following structure prepared by the method of Example ① was dissolved in 120 mℓ of toluene.

This sclareol/toluene solution was then added dropwise to the reaction mixture via the addition funnel over a period of half an hour. The temperature of the reaction mixture at this time may be raised to 55°C.

The reaction mixture was then heated to 80° C. for 2 hours.

The reaction mixture was then worked up by slowly adding 600+d of 5% aqueous sodium hydroxide while cooling with ice.

The toluene layer was separated and washed with saturated aqueous sodium chloride and equal volumes of methyl alcohol and saturated aqueous sodium bicarbonate.

Next, the toluene layer was concentrated to obtain a solid weighing 43.34 g (yield: 86%).

The solid thus obtained has the following structure.

> 1.5 g of this compound was mixed into a solution of 12.0 g potassium hydroxide in 30 mα water. The mixture was placed in a flask equipped with a heater, stirrer, and reflux condenser and refluxed at 80° C. for 3 hours. After 3 hours, the product has a vapor temperature of 162-164°C and a pressure of 15 m
A fairly pure compound having the following structure was obtained by fractional distillation at m/T (g), and the following culture medium was prepared.

Component weight part N) (4N Os 0.2% KH, Po, 0.1% MgS
O4・7H,OO,05% Extracted yeast 0.2% Dextrose 1.0% 500m Micro Little Crypto
coccus 1aurentii, ATCC209
inoculated with 20 isolates. 1 at 25℃, 150rpm
After a week, the resulting product was extracted with 330 mQ of ethyl acetate and the extract was dried over anhydrous sodium sulfate;
. The solvent was distilled on a rotary evaporator. The residue was dissolved in warm hexane and ethyl acetate. When the organic layer was naturally evaporated for 24 hours, pure crystals (350 mg) of the compound with the following structure were obtained.
was gotten.

[Brief explanation of drawings]

Figure 1 shows CrypLococcus albidus
FIG. 2 is a photograph of thin layer chromatography development spots of the product of the example process against pH when using (ATCC 20918). Figure 2 shows the GC of the starting material of Example ①.
- MS spectrum. Figure 3 shows the nuclear magnetic resonance [N
MR] spectrum. FIG. 4 is the nuclear magnetic resonance spectrum shown below. , - 11-meeting - 1-1-see; Fight - 11 things... Stinky eyelids - pulled $4 = 7 -- '1 lh 2 Indication of the incident 1990 Patent Application No. 196449 Title of the invention Biologically pure cultures of microorganisms, lactone production methods,
Relationship with cases involving persons amending diol production methods, compound production methods, and cyclic ether production methods Patent applicant

Claims (1)

[Scope of Claims] (1) A biologically pure culture solution of one microorganism selected from the group consisting of the following bacteria: (a) Cryptococcusalbidus (Sa
ito[Skinnervar. albidus]),
ATCC20918 (b) Bensingtoniaciliata, AT
CC20919(c) Cryptococcuslau
rentii, ATCC20920; and (d)Cry
ptoccusalbidus, ATCC2092
1 (2) Lactone with the following structure ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Under ventilation conditions, the following compounds: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ ▲ Formulas , chemical formulas, tables, etc. ▼ and ▲ mathematical formulas, chemical formulas, tables, etc. ▼ in an aqueous nutrient medium containing one or more compounds selected from the above group of compounds, converting and recovering How to produce as much as possible. This method uses Cr
yptococcusalbidus (Saito[S
kinnervar. albidus]), ATCC2
0918, and Cryptococcus albid.
US, ATCC 20921. (3) By converting a compound selected from the group having the following structure, ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ and ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ In order to produce the following diol in a recoverable amount, Bens
ingtoniaciliata, ATCC20919
, and Cryptococcus laurentii
, ATCC20920. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (4) (a) [1] The pH of the reaction mixture is about 2.5 to about 9.
．． 0 and [2] the temperature of the reaction mixture is about 12°C to about 3°C.
At 3°C, [3] the substrate concentration is about 0.1 g/1l to about 130
g/1l, the following group of compounds: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ ▲ There are mathematical formulas, chemical formulas, tables, etc. There are ▼ under aerated conditions in an aqueous nutrient medium containing one or more compounds of ▼ and an additional carbon source to obtain lactones of the following structure: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ containing a carbon source Cryptococcusalbidus, A.
TCC20918 and Cryptococcus albi
Cultivate a microorganism selected from the group consisting of ATCC 20921 and (b) To produce a compound with the following structure ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ Reduce a lactone with the following structure with a reducing agent, , chemical formulas, tables, etc. ▼ (c) To generate a compound with the following structure ▲ There are mathematical formulas, chemical formulas, tables, etc. d) A method for preparing a compound having the following structure, which is characterized by the step of obtaining a compound having the following structure from the above reaction mixture. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (5) [1] The pH of the reaction mixture is about 2.5 to about 9.0, [2] The temperature of the reaction mixture is about 12℃ to about 33℃, [3] Substrate concentration is about 0.1 g/1L to about 130 g/1
Aerated in an aqueous nutrient medium with one or two compounds and an additional carbon source, from the following groups: A microorganism that converts a substrate consisting of a carbon source and at least one compound selected from the group of compounds mentioned above under conditions to produce the following cyclic ethers in recoverable quantities: , Cryptococcus laure
There are ▲mathematical formulas, chemical formulas, tables, etc. of the following structure, characterized by culturing ntii, ATCC20920▼ Method for preparing cyclic ether.